Ponnusamy Sasikumar

Antidiabetic effect of Merremia emarginata Burm. F. in streptozotocin induced diabetic rats.

OBJECTIVE To investigate the antidiabetic property of Merremia emarginata (M. emarginata) Burm. F. plant in streptozotocin induced diabetic rats. METHODS The dose dependent effects of 28 days oral treatment with methanol extract (100, 200 and 400 mg/kg) from the plant of M. emarginata on blood glucose level, body weight, insulin, total hemoglobin, glycosylated haemoglobin (HbA1C), total protein, serum urea, serum creatinine and carbohydrate metabolizing enzymes were evaluated in streptozotocin induced diabetic rats. Histology of pancreas was also studied. RESULTS A significant decrease in blood glucose, serum urea and serum creatinine and significant increase in body weight, insulin and protein level were observed in diabetic rats treated with M. emarginata. Treatment with M. emarginata resulted in a significant reduction of HbA1C and an increase in total hemoglobin level. The activities of carbohydrate metabolizing enzymes such as hexokinase were significantly increased whereas glucose-6-phosphatase, fructose-1, 6-bisphosphatase were significantly decreased by the administration of M. emarginata in diabetic rats. Histology of diabetic rats treated with M. emarginata showed the pancreatic β-cells regeneration. CONCLUSIONS These findings suggest that M. emarginata has potent antidiabetic activity in streptozotocin induced diabetic rats.

Antihyperglycemic activity and antidiabetic effect of methyl caffeate isolated from Solanum torvum Swartz. fruit in streptozotocin induced diabetic rats

Natural remedies from medicinal plants are considered to be effective and safe alternatives to treat diabetes mellitus. Solanum torvum Swartz. fruit is widely used in the traditional system of medicine to treat diabetes. In the present study methyl caffeate, isolated from S. torvum fruit, was screened for its efficacy in controlling diabetes in animal models. Antihyperglycemic effect of methyl caffeate was studied in normal glucose-fed rats. The effects of oral administration of methyl caffeate (10, 20 and 40 mg/kg) for 28 days on body weight, fasting blood glucose, plasma insulin, hemoglobin, glycated hemoglobin, total protein, hepatic glycogen and carbohydrate metabolism enzymes in streptozotocin induced diabetic rats were investigated. Histological observations in the pancreas and GLUT4 expression in skeletal muscles were also studied. Methyl caffeate at 40 mg/kg significantly prevented the increase in blood glucose level after glucose administration at 60 min in comparison to the hyperglycemic control group. In streptozotocin induced diabetic rats, methyl caffeate produced significant reduction in blood glucose and increased body weight. The levels and/or activities of other biochemical parameters were near normal due to treatment with methyl caffeate. Methyl caffeate treated diabetic rats showed upregulation of GLUT4 and regeneration of β-cells in the pancreas. These results substantiated that methyl caffeate possessed hypoglycemic effect, and it could be developed into a potent oral antidiabetic drug.

In vitro degradation of oxalate by recombinant Lactobacillus plantarum expressing heterologous oxalate decarboxylase

The aim of the present study is to constitutively express heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum and to examine its ability to degrade oxalate in vitro for their future therapy against enteric hyperoxaluria.

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

BackgroundCalcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.ResultsRecombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.ConclusionThe present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Secretion of Biologically Active Heterologous Oxalate Decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 Using Homologous Signal Peptides

Current treatment options for patients with hyperoxaluria and calcium oxalate stone diseases are limited and do not always lead to sufficient reduction in urinary oxalate excretion. Oxalate degrading bacteria have been suggested for degrading intestinal oxalate for the prevention of calcium oxalate stone. Here, we reported a recombinant Lactobacillus plantarum WCFS1 (L. plantarum) secreting heterologous oxalate decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal oxalate. The results showed secretion and functional expression of OxdC protein in L. plantarum driven by signal peptides Lp_0373 and Lp_3050. Supernatant of the recombinant strain containing pLp_0373sOxdC and pLp_3050sOxdC showed OxdC activity of 0.05 U/mg and 0.02 U/mg protein, while the purified OxdC from the supernatant showed specific activity of 18.3 U/mg and 17.5 U/mg protein, respectively. The concentration of OxdC protein in the supernatant was 8–12 μg/mL. The recombinant strain showed up to 50% oxalate reduction in medium containing 10 mM oxalate. In conclusion, the recombinant L. plantarum harboring pLp_0373sOxdC and pLp_3050sOxdC can express and secrete functional OxdC and degrade oxalate up to 50% and 30%, respectively.

Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real‐time PCR experiment shows 94‐fold increased expression of Ll.LtrB intron and 14‐fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E‐0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA− mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA− mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition.

Recombinant Lactic Acid Bacteria Secreting OxdC as a Novel Therapeutic Tool for the Prevention of Kidney Stone Disease

Abstract Kidney stone disease is a dreadful pathological condition, and affects 20% of the population worldwide. The majority of renal stones contain calcium oxalate, and oxalate has vital role in the pathogenesis of renal stone. Humans derive oxalate from endogenous production and increased intake of dietary oxalate. Elevated oxalate levels lead to hyperoxaluria, a major risk factor for recurrent nephrolithiasis. Current treatment options in patients with primary and secondary hyperoxaluria are inadequate, and do not always lead to significant reduction in urinary oxalate excretion. Manipulation of gut flora with the potential probiotic bacteria may have a positive impact on gut oxalate levels, and may decrease oxalate absorption. Several reports documented gut microbes capable to degrade luminal oxalate, and reduce the risk of hyperoxaluria. In addition, the oxalate decarboxylase gene from Bacillus subtilis degrades oxalate into CO2 and formate, and studies have proven that administration of recombinant lactic acid bacteria (LAB) expressing oxalate decarboxylase (OxdC) decreased urinary oxalate excretion, and prevented calcium oxalate stone formation. Thus, potential oxalate degrading probiotic recombinant LAB expressing heterologous oxalate decarboxylase could be beneficial to mitigate hyperoxaluria efficiently by intestinal degradation of dietary oxalate.